抽提纯化及限制性酶切厌氧菌染色体DNA的若干技术问题探讨

Study of Some Technical Problems of Extraction Purification and Enzyme Digestion of Chromosomal DNA of Anaerobes

用几种方法抽提、纯化厌氧菌染色体DNA,并进行酶切分析,探讨有关反应条件及影响因素:(1)不同种属厌氧菌对溶菌酶裂解的敏感性不同,控制适当的反应体积及细菌裂解程度可提高溶菌酶法抽提的效率;对溶菌酶不敏感的厌氧菌可用碱裂解法抽提。(2)使用适当的氯化铯浓度,氯化铯密度梯度超离心法可较好地分离纯化DNA。(3)不同来源及结构的DNA,酶切条件不同;适当的亚精胺浓度可促进酶切反应。

Chromosomal DNAs of different anaerobes were extracted and purified by three methods.Also, restriction enzyme digestion of different DNAs were studied: (1) The efficiency of extracting chr.DNA with lysozyme lysis were not the same in different bacteria because of different sensitivity of bacteria to lysozyme lysis.Proper reaction volume and degree of bacteria lysis were two important factors influencing DNA extraction with this method.(2) Alkaline lysis method was used in those bacteria resistant to lysozyme, which was simple and quick.Chr.DNAs' quality were yield with when reaction time was controlled.(3) Chr.DNAs of anaerobes could be purified by equilibrium centrifugation in CsCl-Ethidium Bromide gradients with a proper concentration of CsCI.(4)Proper concentration of spermidine could promote the restriction enzyme digestion.DNAs with different structure and source require different reaction conditions.